Research by genetic information analysis of various organisms has begun, and information on a number of genes including those of humans and their base sequences, and on the proteins encoded by the gene sequences and sugar chains secondarily produced from these proteins, is being rapidly clarified. Functions of polymers such as genes, proteins and sugar chains whose sequences were clarified can be investigated by various methods. In terms of nucleic acids, major examples of the methods include Northern blotting and Southern blotting, which can be used for investigation of various genes in relation to expression of their biological functions by utilization of various nucleic acid-nucleic acid complementarities. In terms of proteins, representative examples of the methods include Western blotting, which can be used to investigate functions and expression of proteins by utilization of protein-protein reactions.
A known example of the nucleic acid detection method is sandwich hybridization. In sandwich hybridization, a capture probe immobilized on a filter is used. The capture probe is complementary to a first portion of the target nucleic acid. In one stage, the capture probe bound to the filter is exposed to a sample to be investigated for the target nucleic acid sequence, and then exposed to a labeled detection probe complementary to a second portion of the target nucleic acid. The second portion is different from the portion in the target which is complementary to the first probe (i.e., the portions do not overlap with each other) (U.S. Pat. No. 4,486,539, JP 7-75600 A and Sinikka Parkkinen et al., Journal of Medical Virology 20: 279-288 (1986)). That method eliminates the labor required for immobilization of the sample on the filter, and enables selection of a first probe applicable to the support.
Conventionally, detection of a target nucleic acid by sandwich hybridization on a support having a capture probe immobilized thereon required an excessive amount of a detection probe relative to the amount of the target nucleic acid. For example, according to JP 2001-69997 A, 250,000 equivalents of the detection probe with respect to the amount of the target nucleic acid is required. However, use of an excessive amount of the detection probe causes cross-hybridization of the excessive detection probe with capture probes to capture other target nucleic acids (non-specific adsorption), resulting in a low detection sensitivity (S/N ratio), which is problematic.
It could therefore be helpful to provide a method of detecting a target nucleic acid, which enables highly sensitive detection of a target nucleic acid without lowering the detection sensitivity (S/N ratio) even by use of a reduced amount of a detection probe.